GENERAL INTRODUCTION

Bioluminescence is visible light made by living creatures. Such creatures are rare on land but extremely common in the oceans. see more on Marine Bioluminescence by BioScience | Explained here: File:BiolumEN.pdf

Even though Steinbeck, Hemmingway and even Darwin referred to the “phosphorescence of the sea” this is a literary rather than a scientific description. Phosphorescence is the delayed emission of light from a source that has been excited by light. Examples include glow-in-the-dark paints and toys.

Bioluminescence is not the same as fluorescence

As with phosphorescence light emission is stimulated by light not by a chemical reaction. With fluorescence the excitation wavelength is always shorter, that is, higher energy, than the emission wavelength and emission ceases as soon as the excitation source is turned off. Some of this confusion may have arisen because some, but not all, luciferins are fluorescent and a few pass their excitation energy along to fluorescent proteins like GFP.

P. phosphoreum is one of the commonest spoilage organisms of fish such as cod. It is not known to cause disease, but there are reports of people being startled by glowing fish fingers in the fridge!

To isolate glowing bacteria from fish, obtain a freshly-caught seafish or squid. It is very important that the fish has not been frozen or washed in fresh water. It is also better if the fish has not been stored on ice. (That‘s what you usually find everywhere on the Internet. But you can buy almost any saltwater fish fillet from the counter and you will be able to isolate glowing bacteria.)
Place the fish in a container with 3% NaCl solution. The liquid should be deep enough to come half way up the fish.

Cover the container and store the fish for 24 hours at about 12–15 °C. Note: if this temperature is difficult to achieve, place the fish in a fridge at about 4 °C for 48–72 hours.

After incubation, take the container with the fish into a dark room. When your eyes have been adopted to dark, light spots will be visible on
the skin of the fish. Use a sterile toothpick or disposable sterile loop or needle to aseptically transfer the brightest spots onto sterile fish enrichment agar plates. Tip: some people find it useful to use a dim red lamp (e.g., a photographic safety lamp) for this step. Turn the fish away from the lamp so that the glowing colonies are in the shade and therefore visible.

Transfer the cultures to new agar plates every second day if you are incubating them at temperatures around 12 °C or once a week if you are
storing them in a fridge. P. phosphoreum will grow at 4 °C; V. fischeri will not. By selecting the brightest colonies when inoculating, it should be possible to isolate a pure culture.

Fish enrichment medium

Boil 250 g fish meat in 1 l of water.

Add 30 g NaCl and sieve to remove solids.

Add 10 g peptone, 10 ml glycerol and 1 g yeast extract.

Adjust the pH to 7.

Autoclave at 121 °C for 15–20 minutes.

For a solid medium add 15–20 g of agar to every litre of broth.

Safety note
Several species of Vibrio are pathogenic. The chances of inadvertently isolating pathogens in this procedure can be reduced by using at least 3% salt solution and incubating fish and plates at no more than 15 °C. Human pathogens are unlikely to grow under such conditions.

Photorhabdus luminescens

Photorhabdus luminescens (sometimes called Xenorhabdus luminescens) is a bioluminescent microbe and lives in symbiotic relationship with soil entomopathogenic nematodes(family: Heterorhabditidae).

Photorhabdus luminescens bacteria live in Nematodes and produce a toxin that is pathogenic for many insects.
The nematodes infect insect larvae and kill them with the toxin. In the insect larvae, the bacteria excrete next to the fatal toxin also an antibiotic substance to prevent invasion of the insect by bacterial or fungal competitors, and a luminescent protein that causes a faint glow in the infected insect remains.

There have been some reported cases of human infection by Photorhabdus luminescens.
Stories from the American Civil War and the First World War tell of soldiers with wounds that glowed in the dark. Their fluorescing tissues appeared to heal more cleanly and more quickly than the unilluminated wounds of their counterparts. This mysterious phenomenon became known as the „Angel’s Glow“. The afflicted soldiers had no idea that the glow was caused by a beneficial bacterial infection and instead interpreted it as the gift of survival from God, handed to them by angels, hence the name.

Nematodes are used as pesticides in organic farming; so for orchards, for young vines and ornamental plants, against the black vine weevil and the garden chafer.
In Switzerland you can easily buy such nematodes at coop and migros.

Panellus stipticus (Bull.) P. Karst, a naturally bioluminescent tropical fungus, has been studied in vitro for the optimum culture conditions viz culture media, temperature, pH and days of incubation required for luminescence. Temperature and pH affect growth and bioluminescence to a great extent. Glucose-peptone medium has been found to be the best for optimum mycelial growth as well as luminescence. The fungus exhibits luminescence at 20-24°C. The maximum mycelia dry weight (mg/25ml of the basal media) and luminescence observed at pH 4.0. The fungus exhibits luminescence after eight days of incubation at 24°C and pH 4.0, whereas it intensified to maximum after 13 days of incubation (pH 4.0 and temperature 24°C).

tips for Shroom Cultures

GLOWING WOOD

Glowing Wood is a bioluminescence phenomenon caused by a fungus. Some fungi have the ability for bioluminescence and one of them is Armillaria.

Armillaria --> Honey fungus, or Hallimasch (German) or оpenky (Ukrainian) is a genus of parasitic fungi that live on trees and woody shrubs. It includes about 10 species formerly lumped together as Armillaria mellea.
It is known to be one of the largest living organisms, where scientists have estimated a single specimen (of the species Armillaria solidipes) found in Malheur National Forest in Oregon to have been growing for some 2,400 years, covering 3.4 square miles (8.4 km²). Some species of Armillaria are bioluminescent and may be responsible for the phenomena known as „foxfire“ and perhaps „will o‘ the wisp“.
wikipedia.org

A team of Russian and Japanese scientists has now unravelled the previously unknown chemistry responsible for the glowing fungi. The team was able to identify the luciferin precursor as hispidin, a fungal metabolite known to be a strong antioxidant. What makes a fungus able to luminesce isn't hispidin alone; it is the ability to produce the two enzymes necessary for bioluminescence. The researchers were able to extract these two enzymes from the biomass of luminescent fungi:
The first is hispidin-3-hydroxylase, which converts hispidin to the actual luciferin, 3-hydroxylhispidin, by means of a hydroxylation reaction that also involves NADPH.
-> Fungi get the green light: Chemical basis for bioluminescence in glowing fungi uncovered

Historic examples

"Six small pieces of thick glass in the top of the submarine provided natural light.[5] Illumination while submerged was provided by a piece of cork that gave off a fungus-powered bioluminescent foxfire. During trials in November 1775, Bushnell discovered that this illumination failed when the temperature dropped too low. Although repeated requests were made to Benjamin Franklin for possible alternatives, none were forthcoming, and the Turtle was sidelined for the winter."

A clear growing container (shallow containers with lots of surface area)Sanitation is necessary so your batch doesn‘t crash.
REALLY wash out the grow container, make sure there is absolutely no residue left.
Culture flasks -- Sterilized glassware if autoclave is available, otherwise use disposable tissue culture flasks.
If you use sterile media and glassware, your cultures will continue forever; every month pour about 1/4 of the culture into some new medium. If you can‘t maintain sterile culture conditions, the cells will last for a few weeks to a month before bacteria overgrow the culture.

Water

Use purified water as tap water can contain chlorine or other things that might kill your batch.

No tap water, tap water may contain metal ions that are detrimental to algal growth.

Temperature: 22° C (Pyrocystis fusiformis 15°C - 25°C)

Sea Salt from pet or aquarium stores

Light and timer

Indirect natural light is good, but not direct sunlight.

Illumination with cool-white fluorescent lamps

Fluorescent light, either cool white or full spectrum, is also sufficient. Avoid incandescent lamps, as they can overheat the water and kill your algae.

A grow light or an aquarium/terriarium light

Recommendation of Sciento: We finde illumination under 30-40 watt Fluorescent tube or LED lighting ideal and avoid lighting like halogen bulbs or high wattage incandescent bulbs which tend to emit excessive heat.

LUMINOUS WORKSHOPOLOGY

--- the future is bright ---

Why the hell should everything glow?

Phototaxis is a kind of taxis, or locomotory movement, that occurs when a whole organism moves towards or away from stimulus of light.This is advantageous for phototrophic organisms as they can orient themselves most efficiently to receive light for photosynthesis.
(wikipedia.org)

Phototropism is the movement or growth of organisms in response to lights or colors of light (e.g.- the sunflower)
(wikipedia.org)

Heliotropism is the motion of plant parts (flowers or leaves) in response to the direction of the sun. A. P. de Candolle called this phenomenon in any plant heliotropism (1832). It was renamed phototropism in 1892, because it is a response to light rather than to the sun.
(wikipedia.org)

TOWARDS A MINI CITIZEN SCIENCE FESTIVAL IN ZURI

starting with experimentations

Andreas shows the glowing wooden logs they found in the forest around Zurzach

Andreas chopping and preparing wood for the biolum-effect

Can we turn this artist's studio into a micro-GlowLab?

Let's go shopping... Coop in Letzipark will have all we need

... and the rest we get donated from some friends up in ETH Hönggerberg. Thanks to Edmondo for all the fine chemicals and containers

The Mushroom Cultivator, Stamets and Chilton, 1984

Sterile Technique and Agar Culture

Water quality (its pH and mineral content) varies from region to region. If living in an area of questionable water purity, the use of distilled water is advisable. For all practical purposes, however, tap water can be used without harm to the mushroom mycelium. A time may come when balancing pH is important—especially at spore germination or in the culture of exotic species. The pH of media can be altered by adding a drop at a time of 1 molar concentration of hydrochloric acid (HCL) or sodium hydroxide (NaOH). The medium is thoroughly mixed and then measured using a pH meter or pH papers. (One molar HCL has a pH of 0; one molar NaOH has a pH of 12; and distilled water has a pH of 7).

After thoroughly mixing these ingredients, sterilize the medium in a pressure cooker for 30 miutes at 15 psi (equals 103 KPa). (Pressure cookers are a safe and effective means of sterilizing media provided they are operated according to the manufacturer's instructions). A small mouthed vessel is recommended for holding the agar media. If not using a flask specifically manufactured for pouring media, any narrow necked glass bottle will suffice. Be sure to plug its opening with cotton and cover with aluminum foil before inserting into the pressure cooker. The media container should be filled only to 2/3 to 3/4 of its capacity.

Place the media filled container into the pressure cooker along with an adequate amount o water for generating steam. (Usually a 1/2 inch layer of water at the bottom will do). Seal the cooker according to the manufacturer's directions. Place the pressure cooker on a burner and heat until ample steam is being generated. Allow the steam to vent for 4-5 minutes before closing the stop cock. Slowly bring the pressure up to 15 psi and maintain for 1/2 hour. Do not let the temperature of the cooker exceed 250 °F. or else the sugar in the media will caramelize. Media with caramelized sugar inhibits mycelial growth and promotes genetic mutations. A sterilized pot holder or newly laundered cloth should be handy in the sterile lab to aid in removing the media flask from the pressure cooker. While the media is being sterilized, immaculately clean the laboratory.